A major strategy used to fight infection with HIV-1 is the use of protease inhibitors, which are small polypeptide derivatives. Several of these demonstrate high antiretroviral potency to HIV-1 in vitro. However, the low and variable oral bioavailability of protease inhibitors and their inability to penetrate the blood-brain barrier are significant impediments to their use. Initially, metabolism by the cytochrome P450 3A4 isoform was thought to underlie these problems, but recent evidence indicates that multispecific drug export pumps play a major role in limiting drug entry at the gut. Clearly, to be able to modify or bypass these barriers, it is important to identify the most critical physiologically relevant processes.We are using isolated sheets of rat intestine and isolated rat brain capillaries along with radiotracer techniques and confocal microscopy to identify the transporters responsible for reduced protease inhibitor absorption in the gut and protease inhibitor exclusion from the CNS. Initial experiments with a model system and with brain capillaries indicate that both ritonavir and saquinavir interact with two drug export pumps, p-glycoprotein and Mrp2. Of the two protease inhibitors, ritonavir, has the highest affinity for both, being roughly as potent as the best inhibitors of transport yet tested. Plans are to complete identification of relevent transport systems and their contributions to barrier function and to determine how these barriers may be transiently and specifically opened to facilitate treatment.